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Titel (primär) Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex
Autor Ihling, C.; Schmidt, A.; Kalkhof, S.; Schulz, D.M.; Stingl, C.; Mechtler, K.; Haack, M.; Beck-Sickinger, A.G.; Cooper, D.M.F.; Sinz, A.;
Journal / Serie Journal of the American Society for Mass Spectrometry
Erscheinungsjahr 2006
Department PROTEOM;
Band/Volume 17
Heft 8
Sprache englisch;
Abstract For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d(0) and d(4)) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS(2)G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days
ID 2734
dauerhafte UFZ-Verlinkung https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=2734
Ihling, C., Schmidt, A., Kalkhof, S., Schulz, D.M., Stingl, C., Mechtler, K., Haack, M., Beck-Sickinger, A.G., Cooper, D.M.F., Sinz, A. (2006):
Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex
J. Am. Soc. Mass Spectrom. 17 (8), 1100 - 1113